CGH Hybridization: Preparing the Reagents

20X SSC, pH 5.3

Mix thoroughly 66 g 20X SSC in 200 mL purified H2O. Adjust to pH 5.3 at ambient temperature using concentrated HCl and QS to final volume of 250 mL. Store at ambient temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated.

Denaturation solution

Add 49 mL formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2O to a glass Coplin jar and mix thoroughly. Verify that the pH is 7.0 -7.5 by measuring the pH at ambient temperature. Between use, store covered at 4ºC. Discard after 7 days.

Ethanol wash solutions (70%, 85% and 100%)

Dilute 100% ethanol v/v with purified H2O to prepare the wash solutions. Between uses, store covered at ambient temperature. Discard stock solutions after 6 months.

0.4X SSC/0.3% NP-40 Wash Solution

Mix thoroughly 20 mL of 20X SSC with 950 mL purified H2O. Add 3 mL NP-40. Mix thoroughly until NP-40 is dissolved. Adjust pH to 7.0 - 7.5 with NaOH. Add purified H2O to bring final volume to 1 L. Store at ambient temperature. Discard stock solution after 6 months, or if solution appears cloudy or contaminated.

2X SSC/0.1% NP-40 Wash Solution

Add 1 mL NP-40. Adjust pH to 7.0 - 7.5 with NaOH. Add purified H2O to bring final volume to 1 L. Store at ambient temperature. Discard stock soultion after 6 months, or if solution appears cloudy or contaminated.

Test DNA

Direct-label the test (and unlabeled control) DNA with SpectrumGreen^TM dUTP. See the Vysis CGH Nick Translation Kit for the procedure.

Controls 

Positive and negative controls provide comparisons for evaluating the hybridization and interpretation of the CGH data. For a negative control, use normal DNA for both the test and reference DNA (Cat. No.32-804024, 32-802024). The intensity profiles for this experiment should be within the threshold values as determined by image analysis. For a positive control, use test DNA (Cat. No. 32-800227) that is extracted from a cell line with known genetic aberrations that are easy to detect by CGH analysis.

Normal Metaphase CGH Target Slides

Do not pretreat slides. Slides are prepared using standard cytogenetic slide preparation methods that are optimized for CGH. The slides are made from phytohemagglutinin(PHA) stimulated lymphocytes cultured for 48 to 72 hours before thymidine is added to synchronize the cells. The chromosome length is 400 - 550 bands. The lymphocytes are derived from a karyotypically normal male donor.

Preparing the Probe Mix

To produce a hybridization with signal intensities equivalent for both SpectrumGreen^TM and SpectrumRed^TM labeled DNA, the amount of SpectrumGreen labeled DNA is increased.

1. Combine the following in a 1.5 mL microcentrifuge tube:
   10 µL (200 ng) SpectrumGreen (nick translated labeled) test DNA 1 µL (100 ng) SpectrumRed total genomic reference DNA (Cat. No. 32-804023 or 32-804024) 10 µL (10 µg) Human COT-1-1 DNA (Cat. No. 32-800028)
2. Add 1 µL (0.1 volume) 3 M sodium acetate, then add 52.5 µL (2.5volumes) of 100% EtOH to precipitate the DNA. Vortex briefly and place on dry ice for 15 minutes.
3. Centrifuge at 12,000 rpm for 30 minutes at 4°C to pellet the DNA. 
4. Remove the supernatant and dry the pellet for 10 - 15 minutes under a vacuum at ambient temperature.
5. Resuspend the pellet in 3 µL purified H2O and 7µL CGH hybridization buffer. Denature the probe by heating the probe mix for 5 minutes in a 73°C water bath. NOTE: Denature the probe while the slide is being dehydrated (step 3 in Hybridizing the Probe to the Target Metaphase).

Hybridizing the Probe to the Target Metaphase 

1. Mark hybridization areas on the slide using a diamond tipped scribe.
2. Ensure the temperature of the denaturation solution is at 73±1°C. Immerse the slide containing normal metaphase spreads into the solution for 5 minutes.
3. Dehydrate the slide for 1 minute in the 70% ethanol solution, followed by 1 minute in the 85% ethanol solution, and 1 minute in the 100% ethanol solution.
4. Dry the slide by touching the bottom edge to a blotter and wiping the underside with a paper towel.
5. Place the slide on a 45 - 50°C slide warmer to allow remaining EtOH to evaporate.
6. Apply 10 µL of denatured probe mix to the slide. 
7. Immediately apply the coverslip and seal with diluted rubber cement.
8. Place slide in a sealed, humidified box and place in a 37°C incubator for 48 - 72 hours for hybridization.

Washing the Slide 

1. Place the wash tank containing 0.4X SSC/0.3% NP-40 wash solution in a 74±1°C water bath for at least 30 minutes prior to use. Discard after using 1 day. Prepare a second wash tank of 2X SSC/0.1% NP-40 at ambient temperature.
2. Remove the rubber cement seal and the coverslip and immediately place the slide into the 0.4X SSC/0.3% NP-40 wash solution at 74±1°C. Agitate the slide for 1-3 seconds.
3. Repeat step 2 for each slide - do not wash more than four slides at once - and then let the slides stand for 2 minutes.
4.  Place the slide in the 2X SSC/0.1% NP-40 wash solution at ambient temperature. Agitate the slide 1-3 seconds and then let stand for 5 seconds to 1 minute.
5.  Air dry slide in darkness.

Visualizing the Hybridization

Apply 10 µL of DAPI II counterstain and a coverslip to each hybridization location.